Mutations in nuclear genes affecting the activity of complex I in mitochondria are known to cause human disease (neuropathies, myopathies), but the nature of the mutations and how they affect activity is mostly unknown. A long term goal of this proposal is to create mammalian model systems to study mutations in nuclear genes encoding complex I proteins, first in tissue culture and eventually in a mouse model. Five complementation groups of Chinese hamster cell mutants with severe defects in NADH-quinone oxidoreductase (complex I) have been isolated by our laboratory. They represent nuclear genes. Since no such complex exists in yeast, the mammalian cell mutants represent a unique resource and model system for biochemical and molecular genetic studies of the assembly and structure of this mitochondrial complex and its function in electron transport. One of these genes (NDUFA1) encoding the MWFE protein has been identified. This protein had been grouped among the 28 "accessory" proteins of complex I for which no role or function had been defined. We have characterized mutant alleles resulting in a complete loss of activity, and we have other alleles causing variable reduced activities. Among the major goals are: 1) to understand the orientation of the MWFE membrane protein in the inner membrane; 2) to identify the targeting sequence and to understand how this protein is imported into mitochondria; 3) to define the competition between mutant and wild type alleles under conditions where the mutant alleles can behave as dominant-negative mutations. 4) Finally, the preliminary results have already suggested very plausible and rational approaches towards producing transgenic mice and knock-in mice with partial, and possibly tissue specific defects in complex I activity. Such mice promise to become model systems for mitochondrial diseases. 5) Known cDNAs will be tested to identify the defective gene in the other four complementation groups with complex I defects.